RADIATION--DELAYED MUTATION & INSTABILITY IN HUMAN CELLS

拟议工作的目标是确定该现象是否已知 当在体外暴露于电离的人类细胞中发生延迟突变时 辐射。传统的突变检测评估诱导性 在时间的单次快照中发生突变。如果辐射暴露导致 基因组持续不稳定，传统检测方法可能低估了 体细胞可遗传改变的风险。假设是 测试结果是，延迟突变的产生是由于诱导 基因组不稳定。初步研究将使用一位不朽的人， 近二倍体人B淋巴母细胞系，TK6。后续研究 将使用正常的人类淋巴细胞进行实验。以确定是否 延迟致死突变的出现，连续采样的培养效率 将进行照射细胞的检查。确定……的持久性 这种表型，在最初的照射后出现的菌落 为了确定它们的继代培养，将对其进行亚克隆 效率。我们还将确定辐射暴露是否会导致 慢性粒细胞白血病子代特定基因座突变的持续性升高 受辐射的细胞。待检测的候选基因座为次黄嘌呤 磷酸核糖基转移酶(HPRT)基因座，如果可能，还有胸腺嘧啶核苷 激酶基因(Tk)和选定的VNTR和微卫星重复序列基因座。这个 克隆性HPRT中发现的DNA结构改变谱 表现延迟突变表型的突变体将使用 Southern杂交技术或基于聚合酶链式反应的分析以确定它们是否 类似于自发突变，或者他们是否有一个独特的特征 与引起不稳定有关。一项单独的衡量标准 基因组的不稳定性，即扩增CAD基因的能力，也将是 在为上述研究选择的细胞上进行。我们会 进行体细胞杂交实验，以确定 中国人某些受照子代的延迟突变表型 仓鼠卵巢细胞可以被人类染色体抑制。我们还在寻求 要确定延迟突变是否是在 只有当程序性细胞死亡的途径被废除时，人类细胞才会死亡。 这些研究的总体目标是确定辐射是否 暴露会导致人类细胞基因组的持续不稳定， 并披露是否需要有不稳定的倾向 延迟突变表型明显。

The goal of the proposed work is to determine whether the phenomenon known as delayed mutation occurs in human cells exposed, in vitro, to ionizing radiation. Traditional mutation assays evaluate the induction of mutations at a single snapshot in time. If radiation exposure induces a persistent instability in the genome, traditional assays may underestimate the risk of heritable alterations in somatic cells. The hypothesis to be tested is that delayed mutations arise as a result of the induction of genomic instability. Initial studies will be performed using an immortal, near-diploid human B-lymphoblastoid cell line, TK6. Subsequent studies will be performed using normal human lymphocytes. To determine whether delayed lethal mutations arise, serial sampling of the plating efficiency of irradiated cells will be performed. To determine the persistence of this phenotype, colonies arising after the initial post-irradiation plating will be subcloned in order to determine their subcultivation efficiency. We will also determine whether radiation exposure induces a persistent elevation in specific locus mutations in the progeny of irradiated cells. The candidate loci to be tested are the hypoxanthine phosphoribosyltransferase (hprt) locus and, where possible, the thymidine kinase locus (tk) and selected VNTR and microsatellite repeat loci. The spectrum of DNA structural alterations found in clonally derived hprt mutants exhibiting the delayed mutation phenotype will be determined using Southern blotting techniques or PCR-based assays to ascertain whether they resemble spontaneous mutants or whether they have a unique signature associated with the induction of instability. A separate measure of genomic instability, the ability to amplify the CAD gene, will also be performed on the cells chosen for the abovementioned studies. We will carry out somatic cell hybridization experiments to determine whether the delayed mutation phenotype in certain irradiated progeny of Chinese hamster ovary cells can be suppressed by human chromosomes. We also seek to determine whether delayed mutation is a process which is invoked in human cells only when the pathway for programmed cell death is abrogated. The overall goal of these studies is to determine whether radiation exposure induces a persistent instability in the genome of human cells, and to disclose whether a predisposition to instability is required for the delayed mutation phenotype to be evident.